ABSTRACT
AIMS: Proteins of the Polycomb repressive complex 2 (PRC2) are epigenetic gene silencers and are involved in tumour development. Their oncogenic function might be associated with their role in stem cell maintenance. The histone methyltransferase Enhancer of Zeste 2 (EZH2) is a key member of PRC2 function: we have investigated its expression and function in gliomas. METHODS: EZH2 expression was studied in grade II-IV gliomas and in glioma stem-like cells (GSC) by quantitative PCR and immunohistochemistry. Effects of EZH2 down-regulation were analysed by treating GSC with the histone deacetylase (HDAC) inhibitor suberoylanide hydroxamic acid (SAHA) and by shRNA. RESULTS: DNA microarray analysis showed that EZH2 is highly expressed in murine and human GSC. Real-time PCR on gliomas of different grade (n = 66) indicated that EZH2 is more expressed in glioblastoma multiforme (GBM) than in low-grade gliomas (P = 0.0013). This was confirmed by immunohistochemistry on an independent set of 106 gliomas. Treatment with SAHA caused significant up-regulation of PRC2 predicted target genes, GSC disruption and decreased expression of EZH2 and of the stem cell marker CD133. Inhibition of EZH2 expression by shRNA was associated with a significant decrease of glioma proliferation. CONCLUSION: The data suggest that EZH2 plays a role in glioma progression and encourage the therapeutic targeting of these malignancies by HDAC inhibitors.
Subject(s)
Brain Neoplasms/genetics , DNA-Binding Proteins/genetics , Glioma/genetics , Transcription Factors/genetics , Animals , Blotting, Western , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Separation , Chromatin Immunoprecipitation , Disease Progression , Enhancer of Zeste Homolog 2 Protein , Flow Cytometry , Glioma/metabolism , Glioma/pathology , Histone Deacetylase Inhibitors/pharmacology , Humans , Hydroxamic Acids/pharmacology , Immunohistochemistry , Mice , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Oligonucleotide Array Sequence Analysis , Polycomb Repressive Complex 2 , Reverse Transcriptase Polymerase Chain Reaction , Up-Regulation/drug effects , VorinostatABSTRACT
In recent years, the use of dendritic cells (DC), the most powerful antigen presenting cells, has been proposed for the creation of vaccines against gliomas. This approach has been demonstrated to be safe and non-toxic in phase I or I-II trials (2, 3). Immunotherapy plays a central role in the search for new treatments for glioblastoma multiforme (GBM). In particular, several phase I studies have been performed using DC pulsed by GBM proteins as a vaccine for patients with relapsing GBM. The studies demonstrated that DC vaccination is safe and may produce a significant increase in overall survival. As the first step in the preparation of appropriate conditions for a clinical evaluation in Italy, we have performed pre-clinical experiments on immune-competent mice injected intra-cerebrally with syngeneic GL261GBM cells and treated subcutaneously and intra-tumorally with DC loaded with a GL261 homogenate. These results show that vaccination with DC pulsed with a tumor lysate increases considerably survival in mice bearing intracranial glioblastomas and supports the development of DC-based clinical trials for patients with glioblastomas that do not respond to standard therapies.
Subject(s)
Brain Neoplasms/therapy , Cancer Vaccines , Dendritic Cells/immunology , Glioblastoma/therapy , Animals , Bone Marrow Cells/cytology , Brain Neoplasms/immunology , Brain Neoplasms/pathology , Flow Cytometry , Glioblastoma/immunology , Glioblastoma/pathology , Immunotherapy/methods , MiceABSTRACT
During their growth, malignant gliomas interact with the immune system and are able to escape immune reactions. Attempts to instruct the immune system to develop anti-glioma reactions have been partly unsuccessful. Recent advances in the molecular and cellular biology of dendritic cells (DC), however, may increase the chances of preparing effective "vaccines" against these tumours. We show that vaccination with DC pulsed with a tumour lysate considerably increases survival in mice bearing intracranial glioblastomas. These results support the development of DC-based clinical trials for patients with glioblastomas that do not respond to standard therapies.
Subject(s)
Brain Neoplasms/therapy , Cell Transplantation , Dendritic Cells/physiology , Glioma/therapy , Animals , Antigens, CD/metabolism , Brain Neoplasms/immunology , Cancer Vaccines/therapeutic use , Cell Count , Cell Transplantation/methods , Cells, Cultured , Dendritic Cells/transplantation , Glioma/immunology , Mice , Mice, Inbred C57BL , Time FactorsABSTRACT
Melanoma-associated peptides recognized by cytolytic T lymphocytes (CTL) in the context of several histocompatibility leukocyte antigens (HLA) are required for the development of specific immunotherapies. Using a transient transfection assay into COS-7 cells, we identified the gp100/pMel17 melanosomal protein as the shared antigen recognized by three independent CD8+ CTL clones in HLA-A*6801-restricted fashion. This finding was confirmed by the correlation between lack of gp100/pMel17 protein in a number of HLA-A*6801-positive melanomas and their resistance to lysis/cytokine production by the specific effectors. The gp100/pMel17 antigenic epitope was identified based on recognition of subfragments and on a computer-based prediction algorithm. Among a panel of gp100/pMel17-derived synthetic peptides only the 10-mer HTMEVTVYHR (gp100/pMel17182-191) induced tumor necrosis factor (TNF) release by CTL clones when pulsed on suitable target cells whereas both the 10-mer and the shorter 9-mer gp100/pMel17183-191 sensitized the same antigen-pulsed cells to lysis. In conclusion, the identification of the HTMEVTVYHR peptide will extend to HLA-A*6801 melanoma patients the possibility to exploit gp100/pMel17 melanosomal protein for experimental and clinical studies.
